A construction method of rev virus infectious clone expressing green fluorescent envelope fusion protein

A technology of REV virus and fusion protein is applied in the field of construction of infectious clone of REV virus, which can solve problems such as inappropriate insertion sites, and achieve the effect of improving efficiency and simplifying the construction process.

Active Publication Date: 2018-04-20
YANGZHOU UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

Scholars at home and abroad also tried to insert the green fluorescent protein gene into the REV genome to rescue the REV tracer virus expressing green fluorescent protein, but due to the inappropriate insertion site selected, the REV virus expressing the green fluorescent envelope fusion protein has not been successfully obtained so far.

Method used

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  • A construction method of rev virus infectious clone expressing green fluorescent envelope fusion protein
  • A construction method of rev virus infectious clone expressing green fluorescent envelope fusion protein
  • A construction method of rev virus infectious clone expressing green fluorescent envelope fusion protein

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Embodiment

[0028] 1) Design and amplify REV infectious clone linearization vector and EGFP gene fragment primers: amplify the upstream primer of REV infectious clone linearization vector located at position 6112-6141 of the proviral genome of SNV strain; amplify the linearization of LTR containing REV virus The downstream primer of the vector is located at positions 6082-6111 of the proviral genome of the SNV strain; the upstream primer for amplifying the EGFP gene has 15 EGFP gene-specific bases at the 5 end in addition to the 18 EGFP gene-specific bases starting from the EGFP gene ATG Bases that are reverse-complementary to the downstream primers of the linearized vector for amplifying the infectious clone of REV; the downstream primers for amplifying the EGFP gene contain 15 conserved and amplified containing Bases complementary to primers upstream of the REV infectious clone linearized vector. The specific primer sequences are shown in Table 1, which were synthesized by Life Technolo...

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Abstract

The invention relates to a method for constructing an infectious clone of REV virus expressing green fluorescent capsule membrane fusion protein, which is to insert the green fluorescent EGFP gene fragment amplified by specific PCR primers into the N-terminal of the linearized REV virus genome Env gene , using the commercialized recombinase ExnaseTM II without enzyme digestion and ligation reaction, rapid recombination clones in vitro, and then transformed into Escherichia coli for plasmid amplification and identification; and the positive clones were transfected into DF1 cells to rescue and obtain the expression of green fluorescent capsules Membrane fusion protein of REV virus.

Description

technical field [0001] The invention relates to a method for constructing an infectious clone of REV virus expressing green fluorescent envelope fusion protein. This invention can not only obtain the REV tracer virus expressing the green fluorescent envelope fusion protein, which fills the gap at home and abroad; it provides materials for the study of REV virus replication in the body, tissue tropism and its pathogenic mechanism; Viral vectors offer the possibility to express foreign genes. Background technique [0002] Avian reticuloendotheliosis virus (REV) mainly causes a group of syndromes characterized by reticuloendothelial cell hyperplasia, including acute reticulosis, dwarf syndrome and chronic neoplastic hyperplasia of lymphoid tissue and other tissues disease. REV-infected chickens tend to grow stunted, and the elimination rate and mortality increase; and cause immunosuppression of infected chickens, interfere with the immune effect of other poultry disease vacci...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12N15/63
Inventor 叶建强周晓祥秦爱建邵红霞
Owner YANGZHOU UNIV
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